Phosphatase high-throughput screening assay design and selection.

Phosphatases are a heterogeneous group of enzymes catalyzing dephosphorylation of diverse substrates ranging from small organic molecules to large phosphorylated multiprotein complexes. A wide variety of biochemical approaches for measuring phosphatase activity exists. Spectrophotometric methods utilizing artificial chromogenic, fluorogenic, and luminogenic substrates and taking advantage of the optical properties of dephosphorylated products are broadly used by research community. Another major assay type is based on quantitation of the second product of any phosphatase reactions, inorganic phosphate, using a variety of phosphate detection methods. Although, in theory, compatible with any phosphatase substrate, these assays often are unable to provide acceptable high-throughput screening adaptations of native phosphatase reactions. Conversely, phosphatase assays with artificial substrates frequently are incapable to mirror the intricacies of substrate binding and catalysis of the native reaction and, as a result, unable to deliver biologically relevant phosphatase modulators. Utilization of comprehensive phosphatase assay panels, employing honed biochemical assays and cell-based model systems, in conjunction with novel approaches for screening phosphatases may aid in identification of potent, selective, and biologically active phosphatase modulators.